Compositions and methods for detecting and treating esophageal cancer

ABSTRACT

The present invention relates to methods for the in vitro diagnosis of esophageal cancer, and to compositions and methods for the prevention or the treatment of esophageal cancer. Disclosed are said compositions that include an antibody binding to progastrin and disclosed are methods that include the use of an antibody binding to progastrin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of International Patent Application PCT/EP2017/050037,filed on Jan. 2, 2017, and published as WO 2017/114976 on Jul. 6, 2017,which claims priority to European Patent Application 16305139.4, filedon Feb. 5, 2016, and European Patent Application 15307189.9, filed onDec. 31, 2015, all of which are incorporated herein by reference intheir entireties for all purposes.

The present invention relates to the in vitro diagnosis, the preventionand the treatment of cancer, more particularly it relates to methods forthe in vitro diagnosis of esophageal cancer, and to methods andcompositions for the prevention or the treatment of esophageal cancer.Compositions according to the invention comprise a progastrin-bindingmolecule, in particularly an anti-hPG antibody, whereas methodsaccording to the invention comprise the use of a progastrin-bindingmolecule, and particularly to an anti-hPG antibody.

Esophageal cancer arises from esophageal cells, in the tractus betweenthroat and stomach, and has been described as the eighth most commoncancer, affecting more men than women and with rates varying widelyamong countries.

The two most common types of esophageal cancer are esophagealsquamous-cell carcinoma and esophageal adenocarcinoma. A number of morerare subtypes are also known. Squamous-cell carcinoma arises from theepithelial cells of the esophagus, whereas adenocarcinoma arises fromglandular cells present in the lower part of esophagus.

Clinical diagnosis is based on a biopsy, which is usually performedunder endoscopy. The poor outcome of this illness is due in particularto a late diagnosis, due in particular to the absence of early signs andsymptoms. To date, there are no molecular biomarkers that have beentranslated to widespread clinical practice of esophageal cancer (Kaz etal, Cancer Letters, 2014). Treatments depend on the development of thecancer, and usually include surgery, for small-localized tumors, orchemotherapy, possibly in combination with radiation therapy.

Therefore, there is still a need for methods allowing a quick, reliableand cost-effective diagnosis of esophageal cancer, as there is still aneed for new compositions and methods for the prevention or thetreatment of esophageal cancer.

This is the object of the present invention.

The present invention now provides methods for the in vitro diagnosis ofesophageal cancer, wherein said method comprises the detection ofprogastrin in a biological sample from a subject. Preferably, the amountof progastrin in said sample is determined, thus allowing quantificationof progastrin. The present invention also provides a composition for usein the prevention or the treatment of esophageal cancer, wherein saidcomposition comprises an antibody binding to progastrin, and methods forthe prevention or the treatment of esophageal cancer comprising the useof a composition comprising an antibody binding to progastrin, alone orin combination with any other known prevention or therapeutic methodsagainst esophageal cancer.

Human pre-progastrin, a 101 amino acids peptide (Amino acid sequencereference: AAB19304.1), is the primary translation product of thegastrin gene. Progastrin is formed by cleavage of the first 21 aminoacids (the signal peptide) from preprogastrin. The 80 amino acid chainof progastrin is further processed by cleavage and modifying enzymes toseveral biologically active gastrin hormone forms: gastrin 34 (G34) andglycine-extended gastrin 34 (G34-Gly), comprising amino acids 38-71 ofprogastrin, gastrin 17 (G17) and glycine-extended gastrin 17 (G17-Gly),comprising amino acids 55 to 71 of progastrin.

Anti-human progastrin (anti-hPG) monoclonal antibodies and their use fordiagnosis or therapy have been described in the following documents: WO2011/083 088 for colorectal cancer, WO 2011/083 090 for breast cancer,WO 2011/083 091 for pancreatic cancer, WO 2011/116 954 for colorectaland gastrointestinal cancer, and WO 2012/013 609 and WO 2011/083089 forliver pathologies.

The present invention will become more fully understood from thedetailed description given herein and from the accompanying drawings,which are given by way of illustration only and do not limit theintended scope of the invention.

In a first aspect, the present invention relates to a method for the invitro evaluation of a risk of the presence of esophageal cancer, whereinsaid method comprises a step of detecting progastrin in a biologicalsample from a subject. The presence of progestin in the sample indicatesthat there is a risk of the presence of esophageal cancer.

Thus, in a first embodiment, the invention relates to an in vitro methodfor evaluating the risk of the presence of esophageal cancer in asubject, said method comprising the steps of:

-   -   a) contacting a biological sample from said subject with at        least one progastrin-binding molecule, and    -   b) detecting the binding of said progastrin-binding molecule to        progastrin in said sample, wherein said binding indicates a risk        of the presence of esophageal cancer.

The binding of progastrin-binding molecule may be detected by variousassays available to the skilled artisan. Although any suitable means forcarrying out the assays are included within the invention, it can bementioned in particular FACS, ELISA, RIA, western-blot and IHC.

In a preferred embodiment, the method according to the invention for thein vitro evaluation of a risk of the presence of esophageal cancer in asubject, comprises the steps of:

-   -   a) contacting said biological sample with at least one        progastrin-binding molecule,    -   b) determining the concentration of progastrin in said        biological sample, wherein a concentration of progastrin of at        least 10 μM in said biological sample is indicative of a risk of        the presence of esophageal cancer.

Once the concentration of progastrin present in the sample isdetermined, the result can be compared with those of control sample(s),which is (are) obtained in a manner similar to the test samples but fromindividual(s)s known not to suffer from a esophageal cancer. If theconcentration of progastrin is significantly more elevated in the testsample, it may be concluded that there is an increased likelihood thatthe subject from whom it was derived has an esophageal cancer.

Thus, in a more preferred embodiment, the method of the inventioncomprises the further steps of:

-   -   c) determining a reference concentration of progastrin in a        reference sample,    -   d) comparing the concentration of progastrin in said biological        sample with said reference concentration of progastrin,    -   e) evaluating, from the comparison of step d), the risk of the        presence of esophageal cancer

According to another aspect, the invention relates to an in vitro methodfor diagnosing esophageal cancer in a subject, said method comprisingthe steps of:

-   -   a) contacting a biological sample from said subject with at        least one progastrin-binding molecule, and    -   b) detecting the binding of said progastrin-binding molecule to        progastrin in said sample, wherein said binding indicated the        presence of esophageal cancer in said subject.

In a preferred embodiment, the present invention relates to a method forthe in vitro diagnosis of esophageal cancer in a subject comprising thesteps of:

-   -   a) contacting said biological sample with at least one        progastrin-binding molecule,    -   b) determining the level or concentration of progastrin in said        biological sample, wherein a concentration of progastrin of at        least 10 pM in said biological sample is indicative of the        presence of esophageal cancer in said subject.

In a more particular embodiment of a method according to the invention,a concentration of progastrin of at least 10 pM, at least 20 pM, atleast 30 pM, in said biological sample is indicative of the presence ofesophageal cancer in said subject.

In a more preferred embodiment, the method of the invention comprisesthe further steps of:

-   -   c) determining a reference concentration of progastrin in a        reference sample,    -   d) comparing the concentration of progastrin in said biological        sample with said reference level or concentration of progastrin,    -   e) diagnosing, from the comparison of step d), the presence of        esophageal cancer.

According to another aspect, the invention relates to an in vitro methodfor diagnosing metastasized esophageal cancer in a subject, said methodcomprising the steps of:

-   -   a) contacting a biological sample from said subject with at        least one progastrin-binding molecule, and    -   b) detecting the binding of said progastrin-binding molecule to        progastrin in said sample, wherein said binding indicates the        presence of metastasized esophageal cancer in said subject.

In a preferred embodiment, the present invention relates to a method forthe in vitro diagnosis of metastasized esophageal cancer in a subject,from a biological sample of said subject, comprising the steps of:

-   -   a) contacting said biological sample with at least one        progastrin-binding molecule,    -   b) determining by a biochemical assay the level or concentration        of progastrin in said biological sample, wherein a concentration        of progastrin of at least 10 pM higher in said biological sample        is indicative of the presence of metastasized esophageal cancer        in said subject.

In a more particular embodiment of a method according to the invention,a concentration of progastrin of at least 10 pM, at least 20 pM, atleast 30 pM, at least 40 pM or at least 50 pM in said biological sampleis indicative of the presence of metastasized esophageal cancer in saidsubject.

In a more preferred embodiment, the method of the invention comprisesthe further steps of:

-   -   c) determining a reference concentration of progastrin in a        reference sample,    -   d) comparing the concentration of progastrin in said biological        sample with said reference level or concentration of progastrin,    -   e) diagnosing, from the comparison of step d), the presence of        metastasized esophageal cancer.

In a particular embodiment, the present invention relates to a methodfor the in vitro diagnosis of esophageal cancer in a subject, comprisingthe determination of the concentration of progastrin in a biologicalsample and comparing said value obtained to the concentration ofprogastrin in a reference sample.

In a more particular embodiment, in a method for the diagnosis ofesophageal cancer according to the present invention, the biologicalsample of said subject is contacted with at least one progastrin-bindingmolecule, wherein said progastrin-binding molecule is an antibody, or anantigen-binding fragment thereof.

The expression “evaluation of a risk of the presence of esophagealcancer in a subject” designates the determination of a relativeprobability for a given subject to suffer from esophageal cancer, whencompared to a reference subject or value. A method according to theinvention represents a tool in the evaluation of said risk, incombination with other methods or indicators such as clinicalexamination, biopsy and determination of the level of a known biomarkerof esophageal cancer.

According a particular embodiment, the present invention relates to anin vitro diagnosis method of esophageal cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of esophageal cancer. Clinical symptoms of esophageal cancerinclude weight loss, painfull or difficult swallowing, cough,indigestion and heartburn.

According another particular embodiment, the present invention relatesto an in vitro diagnosis method of esophageal cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of cancer and/or of metastasis.

Therefore, a method for the in vitro diagnosis of esophageal cancer,according to the present invention can be considered as a tool within adiagnosis process.

In a more particular embodiment, the present invention relates to amethod for the in vitro diagnosis of esophageal cancer in a subject,comprises the determination of the concentration of progastrin in saidbiological sample and the determination of a known biomarker ofesophageal cancer.

The term “progastrin” designates the mammalian progastrin peptide, andparticularly human progastrin. For the avoidance of doubt, without anyspecification, the expression “human progastrin” refers to the human PGof sequence SEQ ID No. 1. Human progastrin comprises notably aN-terminus and a C-terminus domains which are not present in thebiologically active gastrin hormone forms mentioned above. Preferably,the sequence of said N-terminus domain is represented by SEQ ID NO. 2.In another preferred embodiment, the sequence of said C-terminus domainis represented by SEQ ID NO. 3.

The determination of the concentration of progastrin, in a methodaccording to the invention, is performed by any method known by oneskilled in the art of biochemistry.

Preferably, determining the levels of progastrin in a sample includescontacting said sample with a progastrin-binding molecule and measuringthe binding of said progastrin-binding molecule to progastrin.

When expression levels are measured at the protein level, it may benotably performed using specific progastrin-binding molecules, such ase.g., antibodies, in particular using well known technologies such ascell membrane staining using biotinylation or other equivalenttechniques followed by immunoprecipitation with specific antibodies,western blot, ELISA or ELISPOT, enzyme-linked immunosorbant assays(ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC),immunofluorescence (IF), antibodies microarrays, or tissue microarrayscoupled to immunohistochemistry. Other suitable techniques include FRETor BRET, single cell microscopic or histochemistry methods using singleor multiple excitation wavelength and applying any of the adaptedoptical methods, such as electrochemical methods (voltametry andamperometry techniques), atomic force microscopy, and radio frequencymethods, e.g. multipolar resonance spectroscopy, confocal andnon-confocal, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry), cell ELISA, flow cytometry, radioisotopic,magnetic resonance imaging, analysis by polyacrylamide gelelectrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; LiquidChromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)). Allthese techniques are well known in the art and need not be furtherdetailed here. These different techniques can be used to measure theprogastrin levels.

Said method may in particular be chosen among: a method based onimmuno-detection, a method based on western blot, a method based on massspectrometry, a method based on chromatography, and a method based onflow cytometry. Although any suitable means for carrying out the assaysare included within the invention, methods such as FACS, ELISA, RIA,western-blot and IHC are particularly useful for carrying out the methodof the invention.

In a more particular embodiment, a method for the in vitro diagnosis ofesophageal cancer according to the invention comprises contacting abiological sample from a subject with a progastrin binding moleculeusing an immunoenzymatic assay, preferably based on techniques chosenamong RIA and ELISA.

A “biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of anesophageal cancer protein, polynucleotide or transcript. Such a samplemust allow for the determination of the expression levels of progastrin.Progastrin is known to be a secreted protein. Preferred biologicalsamples for the determination of the level of the progastrin proteinthus include biological fluids. A “biological fluid” as used hereinmeans any fluid that includes material of biological origin. Preferredbiological fluids for use in the present invention include bodily fluidsof an animal, e.g. a mammal, preferably a human subject. The bodilyfluid may be any bodily fluid, including but not limited to blood,plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat andurine. Preferably, said preferred liquid biological samples includesamples such as a blood sample, a plasma sample, or a serum sample. Morepreferably, the biological sample is a blood sample. Indeed, such ablood sample may be obtained by a completely harmless blood collectionfrom the patient and thus allows for a non-invasive assessment of therisks that the subject will develop a tumor.

A “biological sample” as used herein also includes a solid cancer sampleof the patient to be tested, when the cancer is a solid cancer. Suchsolid cancer sample allows the skilled person to perform any type ofmeasurement of the level of the biomarker of the invention. In somecases, the methods according to the invention may further comprise apreliminary step of taking a solid cancer sample from the patient. By a“solid cancer sample”, it is referred to a tumor tissue sample. Even ina cancerous patient, the tissue which is the site of the tumor stillcomprises non tumor healthy tissue. The “cancer sample” should thus belimited to tumor tissue taken from the patient. Said “cancer sample” maybe a biopsy sample or a sample taken from a surgical resection therapy.

A biological sample is typically obtained from a eukaryotic organism,most preferably a mammal, or a bird, reptile, or fish. Indeed, a“subject” which may be subjected to the method described herein may beany of mammalian animals including human, dog, cat, cattle, goat, pig,swine, sheep and monkey; or a bird; reptile; or fish. Preferably, asubject is a human being; a human subject may be known as a “patient”.

By “obtaining a biological sample,” it is herein meant to obtain abiological sample for use in methods described in this invention. Mostoften, this will be done by removing a sample of cells from an animal,but can also be accomplished by using previously isolated cells (e.g.,isolated by another person, at another time, and/or for anotherpurpose), or by performing the methods of the invention in vivo.Archival tissues, having treatment or outcome history, will beparticularly useful.

This sample may be obtained and if necessary prepared according tomethods known to a person skilled in the art. In particular, it is wellknown in the art that the sample should be taken from a fasting subject.

The determination of the concentration of progastrin relates to thedetermination of the quantity of progastrin in known volume of a sample.The concentration of progastrin may be expressed relatively to areference sample, for example as a ratio or a percentage. Theconcentration may also be expressed as the intensity or localization ofa signal, depending on the method used for the determination of saidconcentration. Preferably, the concentration of a compound in a sampleis expressed after normalization of the total concentration of relatedcompounds in said sample, for example the level or concentration of aprotein is expressed after normalization of the total concentration ofproteins in the sample.

Preferably, the risk that said subject suffers from esophageal cancer isdetermined by comparing the level of progastrin measured in saidbiological sample with a reference level.

The term “reference level”, as used herein, refers to the expressionlevel of the esophageal cancer marker under consideration, i.e.progastrin, in a reference sample. A “reference sample”, as used herein,means a sample obtained from subjects, preferably two or more subjects,known to be free of the disease or, alternatively, from the generalpopulation. The suitable reference expression levels of progastrin canbe determined by measuring the expression levels of said marker inseveral suitable subjects, and such reference levels can be adjusted tospecific subject populations. The reference value or reference level canbe an absolute value; a relative value; a value that has an upper or alower limit; a range of values; an average value; a median value, a meanvalue, or a value as compared to a particular control or baseline value.A reference value can be based on an individual sample value such as,for example, a value obtained from a sample from the subject beingtested, but at an earlier point in time. The reference value can bebased on a large number of samples, such as from population of subjectsof the chronological age matched group, or based on a pool of samplesincluding or excluding the sample to be tested.

Advantageously, a “reference level” is a predetermined progastrin level,obtained from a biological sample from a subject with a known particularstatus as regards cancer. In particular embodiments, the reference levelused for comparison with the test sample in step (b) may have beenobtained from a biological sample from a healthy subject, or from abiological sample from a subject suffering from cancer; it is understoodthat the reference expression profile can also be obtained from a poolof biological samples of healthy subjects or from a pool of samples fromsubjects having cancer.

In an particular embodiment of the method of the invention, thereference sample is collected from subjects exempt from any cancer, andpreferably from any pathology. It is to be understood that, according tothe nature of the biological sample collected from a patient, thereference sample will be a biological sample of the same nature of saidbiological sample.

The level of progastrin is determined in the present method bydetermining the amount of progastrin which is bound by aprogastrin-binding molecule, preferably by an antibody recognisingprogastrin.

By “progastrin-binding molecule”, it is herein referred to any moleculethat binds progastrin, but does not bind gastrin-17 (G17), gastrin-34(G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extendedgastrin-34 (G34-Gly). The progastrin-binding molecule of the presentinvention may be any progastrin-binding molecule, such as, for instance,an antibody molecule or a receptor molecule. Preferably, theprogastrin-binding molecule is an anti-progastrin antibody or anantigen-binding fragment thereof.

According a particular embodiment, the present invention relates to anin vitro diagnosis method of esophageal cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of esophageal cancer.

According another particular embodiment, the present invention relatesto an in vitro diagnosis method of esophageal cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of cancer and/or of metastasis.

By “binding”, “binds”, or the like, it is intended that the antibody, orantigen binding fragment thereof, forms a complex with an antigen which,under physiologic conditions, is relatively stable. Methods fordetermining whether two molecules bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. In a particular embodiment, said antibody, orantigen-binding fragment thereof, binds to progastrin with an affinitythat is at least two-fold greater than its affinity for binding to anon-specific molecule such as BSA or casein. In a more particularembodiment, said antibody, or antigen-binding fragment thereof, bindsonly to progastrin.

In a particular embodiment, in a method for the diagnosis of esophagealcancer according to the invention, a biological sample from the subjectis contact with at least one progastrin-binding molecule, wherein theaffinity of said molecule for progastrin is at least 100 nM, at least 90nM, at least 80 nM, at least 70 nM, at least 60 nM, at least 50 nM, atleast 40 nM, at least 30 nM, at least 20 nM, at least 10 nM, at least 5nM, at least 100 pM, at least 10 pM, or at least 1 pM, as determined bya method such as above-described.

In a particular embodiment, the present invention relates to a methodfor the diagnosis of esophageal cancer, comprising the detection of theconcentration of progastrin in a biological sample from a subject,wherein said biological sample is contacted with an anti-hPG antibody,or an antigen-binding fragment thereof.

The term “antibody” as used herein is intended to include polyclonal andmonoclonal antibodies. An antibody (or “immunoglobulin”) consists of aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain comprises aheavy chain variable region (or domain) (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2 and CH3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region comprisesone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions” (CDR) or “hypervariable regions”, which are primarilyresponsible for binding an epitope of an antigen, and which areinterspersed with regions that are more conserved, termed frameworkregions (FR). Method for identifying the CDRs within light and heavychains of an antibody and determining their sequence are well known tothe skilled person. For the avoidance of doubt, in the absence of anyindication in the text to the contrary, the expression CDRs means thehypervariable regions of the heavy and light chains of an antibody asdefined by IMGT, wherein the IMGT unique numbering provides astandardized delimitation of the framework regions and of thecomplementary determining regions, CDR1-IMGT: 27 to 38, CDR2.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cystein 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

Each VH and VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g. effector cells) and the first component (Clq) ofthe classical complement system. Antibodies can be of different isotypes(namely IgA, IgD, IgE, IgG or IgM).

In a more particular embodiment, said progastrin-binding antibody, or anantigen-binding fragment thereof, is selected from the group consistingof: polyclonal antibodies, monoclonal antibodies, chimeric antibodies,single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2antibodies, IgG3 antibodies, IgG4 antibodies and IgM antibodies.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

The term “monoclonal antibody” designates an antibody arising from anearly homogeneous antibody population, wherein population comprisesidentical antibodies except for a few possible naturally-occurringmutations which can be found in minimal proportions. A monoclonalantibody arises from the growth of a single cell clone, such as ahybridoma, and is characterized by heavy chains of one class andsubclass, and light chains of one type.

By the expression “antigen-binding fragment” of an antibody, it isintended to indicate any peptide, polypeptide, or protein retaining theability to bind to the target (also generally referred to as antigen) ofthe said antibody, generally the same epitope, and comprising an aminoacid sequence of at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least 80 contiguous amino acid residues, at least 90 contiguous aminoacid residues, at least 100 contiguous amino acid residues, at least 125contiguous amino acid residues, at least 150 contiguous amino acidresidues, at least 175 contiguous amino acid residues, or at least 200contiguous amino acid residues, of the amino acid sequence of theantibody.

In a particular embodiment, the said antigen-binding fragment comprisesat least one CDR of the antibody from which it is derived. Still in apreferred embodiment, the said antigen binding fragment comprises 2, 3,4 or 5 CDRs, more preferably the 6 CDRs of the antibody from which it isderived.

The “antigen-binding fragments” can be selected, without limitation, inthe group consisting of Fv, scFv (sc for single chain), Fab, F(ab′)₂,Fab′, scFv-Fc fragments or diabodies, or fusion proteins with disorderedpeptides such as XTEN (extended recombinant polypeptide) or PAS motifs,or any fragment of which the half-life time would be increased bychemical modification, such as the addition of poly(alkylene) glycolsuch as poly(ethylene) glycol (“PEGylation”) (pegylated fragments calledFv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG or Fab′-PEG) (“PEG” forPoly(Ethylene) Glycol), or by incorporation in a liposome, saidfragments having at least one of the characteristic CDRs of the antibodyaccording to the invention. Preferably, said “antigen-binding fragments”will be constituted or will comprise a partial sequence of the heavy orlight variable chain of the antibody from which they are derived, saidpartial sequence being sufficient to retain the same specificity ofbinding as the antibody from which it is descended and a sufficientaffinity, preferably at least equal to 1/100, in a more preferred mannerto at least 1/10, of the affinity of the antibody from which it isdescended, with respect to the target.

In another particular embodiment, in a method for the diagnosis ofesophageal cancer according to the invention, a biological sample from asubject is contacted with an antibody binding to progastrin, whereinsaid antibody has been obtained by an immunization method known by aperson skilled in the art, wherein using as an immunogen a peptide whichamino acid sequence comprises the totality or a part of the amino-acidsequence of progastrin. More particularly, said immunogen comprises apeptide chosen among:

-   -   a peptide which amino acid sequence comprises, or consists of,        the amino acid sequence of full length progastrin, and        particularly full length human progastrin of SEQ ID NO 1,    -   a peptide which amino acid sequence corresponds to a part of the        amino acid sequence of progastrin, and particularly full length        human progastrin of SEQ ID NO 1,    -   a peptide which amino acid sequence corresponds to a part or to        the whole amino acid sequence of the N-terminal part of        progastrin, and in particular peptides comprising, or consisting        of, the amino acid sequence: SWKPRSQQPDAPLG (SEQ ID NO 2), and    -   a peptide which amino acid sequence corresponds to a part or to        the whole amino acid sequence of the C-terminal part of        progastrin, and in particular peptides comprising, or consisting        of, the amino acid sequence: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID        NO 3),    -   a peptide which amino acid sequence corresponds to a part of the        amino acid sequence of the C-terminal part of progastrin, and in        particular peptides comprising the amino acid sequence        FGRRSAEDEN (SEQ ID NO 40) corresponding to amino acids 71-80 of        progastrin

The skilled person will realize that such immunization may be used togenerate either polyclonal or monoclonal antibodies, as desired. Methodsfor obtaining each of these types of antibodies are well known in theart. The skilled person will thus easily select and implement a methodfor generating polyclonal and/or monoclonal antibodies against any givenantigen.

Examples of monoclonal antibodies which were generated by using animmunogen comprising the amino-acid sequence “SWKPRSQQPDAPLG”,corresponding to the amino acid sequence 1-14 of human progastrin(N-terminal extremity) include, but are not restricted to, monoclonalantibodies designated as: mAb3, mAb4, mAb16, and mAb19 and mAb20, asdescribed in the following Table 1 to Table 4. Other monoclonalantibodies have been described, although it is not clear whether theseantibodies actually bind progastrin (WO 2006/032980). Experimentalresults of epitope mapping show that mAb3, mAb4, mAb16, and mAb19 andmAb20 do specifically bind an epitope within said hPG N-terminal aminoacid sequence. Polyclonal antibodies recognizing specifically an epitopewithin the N-terminus of progastrin represented by SEQ ID NO. 2, havebeen described in the art (see e.g, WO 2011/083088).

TABLE 1 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o)6B5B11C10 mAb3 VH CDR 1 GYIFTSYW SEQ ID N^(o) 4 VH CDR 2 FYPGNSDS SEQ IDN^(o) 5 VH CDR 3 TRRDSPQY SEQ ID N^(o) 6 VL CDR 1 QSIVHSNG SEQ NTY IDN^(o) 7 VL CDR 2 KVS SEQ ID N^(o) 8 VL CDR 3 FQGSHVPFT SEQ ID N^(o) 9

TABLE 2 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o) 20D2C3G2mAb4 VH CDR 1 GYTFSSW SEQ ID N^(o) 10 VH CDR 2 FLPGSGST SEQ ID N^(o) 11VH CDR 3 ATDGNYD SEQ WFAY ID N^(o) 12 VL CDR 1 QSLVHSSG SEQ VTY ID N^(o)13 VL CDR 2 KVS SEQ ID N^(o) 14 VL CDR 3 SQSTHVPPT SEQ ID N^(o) 15

TABLE 3 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o) 1E9D9B6mAb16 VH CDR 1 GYTFTSYY SEQ ID N^(o) 16 VH CDR 2 INPSNGGT SEQ ID N^(o)17 VH CDR 3 TRGGYYPF SEQ DY ID N^(o) 18 VL CDR 1 QSLLDSDG SEQ KTY IDN^(o) 19 VL CDR 2 LVS SEQ ID N^(o) 20 VL CDR 3 WQGTHSPYT SEQ ID N^(o) 21

TABLE 4 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o) 1B3B4F11mAb19 VH CDR 1 GYSITSDYA SEQ ID N^(o) 22 VH CDR 2 ISFSGYT SEQ ID N^(o)23 VH CDR 3 AREVNYGD SEQ SYHFDY ID N^(o) 24 VL CDR 1 SQHRTYT SEQ IDN^(o) 25 VL CDR 2 VKKDGSH SEQ ID N^(o) 26 VL CDR 3 GVGDAIKG SEQ QSVFV IDN^(o) 27

Examples of monoclonal antibodies that can be generated by using animmunogen comprising the amino-acid sequence“QGPWLEEEEEAYGWMDFGRRSAEDEN”, (C-terminal part of progastrin)corresponding to the amino acid sequence 55-80 of human progastrininclude, but are not restricted to antibodies designated as: mAb8 andmAb13 in the following Table 5 and 6. Experimental results of epitopemapping show that mAb13 do specifically bind an epitope within said hPGC-terminal amino acid sequence.

TABLE 5 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o) 1C10D3B9mAb8 VH CDR 1 GFTFTTYA SEQ ID N^(o) 28 VH CDR 2 ISSGGTYT SEQ ID N^(o) 29VH CDR 3 ATQGNYSL SEQ DF ID N^(o) 30 VL CDR 1 KSLRHTKGI SEQ TF ID N^(o)31 VL CDR 2 QMS SEQ ID N^(o) 32 VL CDR 3 AQNLELPLT SEQ ID N^(o) 33

TABLE 6 Hybridoma Amino acid SEQ deposit mAb sequences ID N^(o) 2C6C3C7mAb13 VH CDR 1 GFIFSSYG SEQ ID N^(o) 34 VH CDR 2 INTFGDRT SEQ ID N^(o)35 VH CDR 3 ARGTGTY SEQ ID N^(o) 36 VL CDR 1 QSLLDSD SEQ GKTY ID N^(o)37 VL CDR 2 LVS SEQ ID N^(o) 38 VL CDR 3 WQGTHF SEQ PQT ID N^(o) 39

Other examples include anti-hPG monoclonal and/or polyclonal antibodiesgenerated by using an immunogen comprising an amino acid sequence of SEQID NO 40.

In a more particular embodiment, in a method according to the inventionsaid biological sample is contacted with an anti-hPG antibody orantigen-binding fragment thereof, wherein said anti-hPG antibody ischosen among N-terminal anti-hPG antibodies and C-terminal anti-hPGantibodies.

The terms “N-terminal anti-hPG antibodies” and “C-terminal anti-hPGantibodies” designate antibodies binding to an epitope comprising aminoacids located in the N-terminal part of hPG or to an epitope comprisingamino acids located in the C-terminal part of hPG, respectively.Preferably, the term “N-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 2. In another preferredembodiment, the term “C-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 3.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those amino acids that directly contribute to the affinity ofthe interaction. Epitopes may also be conformational. In certainembodiments, epitopes may include determinants that are chemicallyactive surface groupings of molecules such as amino acids, sugar sidechains, phosphoryl groups, or sulfonyl groups, and, in certainembodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. Thedetermination of the epitope bound by an antibody may be performed byany epitope mapping technique, known by a man skilled in the art. Anepitope may comprise different amino acids, which located sequentiallywithin the amino acid sequence of a protein. An epitope may alsocomprise amino acids, which are not located sequentially within theamino acid sequence of a protein.

In a particular embodiment, said antibody is a monoclonal antibodychosen in the group consisting of:

-   -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 4, 5        and 6, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID NO 4, 5 and 6, respectively, and a light chain        comprising at least one, preferentially at least two,        preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid        sequences SEQ ID NO 7, 8 and 9, respectively, or sequences with        at least 80%, preferably 85%, 90%, 95% and 98% identity after        optimal alignment with sequences SEQ ID NO 7, 8 and 9,        respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 10,        11 and 12, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 10, 11 and 12, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 13, 14 and 15, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 13, 14        and 15, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 16,        17 and 18, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 16, 17 and 18, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 19, 20 and 21, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 19, 20        and 21, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 22,        23 and 24, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 22, 23 and 24, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 25, 26 and 27, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 25, 26        and 27, respectively,    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially at least        three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ        ID NO 28, 29 and 30, respectively, or sequences with at least        80%, preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 28, 29 and 30, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 31, 32 and 33, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 31, 32        and 33, respectively, and    -   A monoclonal antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 34,        35 and 36, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 34, 35 and 36, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 37, 38 and 39, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 37, 38        and 39, respectively.

As used herein, the “percentage identity” or “% identity” between twosequences of nucleic acids or amino acids means the percentage ofidentical nucleotides or amino acid residues between the two sequencesto be compared, obtained after optimal alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly along their length. The comparison of two nucleicacid or amino acid sequences is traditionally carried out by comparingthe sequences after having optimally aligned them, said comparison beingable to be conducted by segment or by using an “alignment window”.Optimal alignment of the sequences for comparison can be carried out, inaddition to comparison by hand, by means of methods known by a manskilled in the art.

For the amino acid sequence exhibiting at least 80%, preferably 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated.

In another particular embodiment, the antibody used in the method of theinvention is a humanised antibody.

As used herein, the expression “humanized antibody” means an antibodythat contains CDR regions derived from an antibody of nonhuman origin,the other parts of the antibody molecule being derived from one orseveral human antibodies. In addition, some of the skeleton segmentresidues (called FR for framework) can be modified to preserve bindingaffinity, according to techniques known by a man skilled in the art(Jones et al., Nature, 321:522-525, 1986). The goal of humanisation is areduction in the immunogenicity of a xenogenic antibody, such as amurine antibody, for introduction into a human, while maintaining thefull antigen binding affinity and specificity of the antibody.

The humanized antibodies of the invention or fragments of same can beprepared by techniques known to a person skilled in the art (such as,for example, those described in the documents Singer et al., J. Immun.,150:2844-2857, 1992). Such humanized antibodies are preferred for theiruse in methods involving in vitro diagnoses or preventive and/ortherapeutic treatment in vivo. Other humanization techniques, are alsoknown to a person skilled in the art. Indeed, Antibodies can behumanized using a variety of techniques including CDR-grafting (EP 0 451261; EP 0 682 040; EP 0 939 127; EP 0 566 647; U.S. Pat. Nos. 5,530,101;6,180,370; 5,585,089; 5,693,761; 5,639,641; 6,054,297; 5,886,152; and5,877,293), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. etal., 1994, Protein Engineering 7(6): 805-814; Roguska M. A. et al.,1994, Proc. Natl. Acad. ScL U.S.A., 91:969-973), and chain shuffling(U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety ofmethods known in the art including phage display methods. See also U.S.Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; andinternational patent application publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741.

In a more particular embodiment, said antibody is a humanized antibodyselected in the group consisting of:

-   -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 4, 5        and 6, respectively, or sequences with at least 80%, preferably        85%, 90%, 95% and 98% identity after optimal alignment with        sequences SEQ ID NO 4, 5 and 6, respectively, and a light chain        comprising at least one, preferentially at least two,        preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid        sequences SEQ ID NO 7, 8 and 9, respectively, or sequences with        at least 80%, preferably 85%, 90%, 95% and 98% identity after        optimal alignment with sequences SEQ ID NO 7, 8 and 9,        respectively,    -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 10,        11 and 12, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 10, 11 and 12, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 13, 14 and 15, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 13, 14        and 15, respectively,    -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 16,        17 and 18, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 16, 17 and 18, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 19, 20 and 21, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 19, 20        and 21, respectively,    -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 22,        23 and 24, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 22, 23 and 24, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 25, 26 and 27, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 25, 26        and 27, respectively,    -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 28,        29 and 30, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 28, 29 and 30, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 31, 32 and 33, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 31, 32        and 33, respectively, and    -   A humanized antibody comprising a heavy chain comprising at        least one, preferentially at least two, preferentially three, of        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 34,        35 and 36, respectively, or sequences with at least 80%,        preferably 85%, 90%, 95% and 98% identity after optimal        alignment with sequences SEQ ID NO 34, 35 and 36, respectively,        and a light chain comprising at least one, preferentially at        least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of        amino acid sequences SEQ ID NO 37, 38 and 39, respectively, or        sequences with at least 80%, preferably 85%, 90%, 95% and 98%        identity after optimal alignment with sequences SEQ ID NO 37, 38        and 39, respectively,

wherein said antibody also comprises constant regions of the light-chainand the heavy-chain derived from a human antibody.

In a first embodiment, a method according to the invention comprisescontacting a biological sample with an anti-hPG antibody binding to anepitope of hPG, wherein said epitope is located within the C-terminalpart of hPG or to an epitope located within the N-terminal part of hPG.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with an anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the N-terminalpart of progastrin chosen among an amino acid sequence corresponding toamino acids 10 to 14 of hPG, amino acids 9 to 14 of hPG, amino acids 4to 10 of hPG, amino acids 2 to 10 of hPG and amino acids 2 to 14 of hPG,wherein the amino acid sequence of hPG is SEQ ID NO 1.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with an anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the C-terminalpart of progastrin, chosen among an amino acid sequence corresponding toamino acids 71 to 74 of hPG, amino acids 69 to 73 of hPG, amino acids 71to 80 of hPG (SEQ ID NO 40), amino acids 76 to 80 of hPG, and aminoacids 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQ IDNO 1.

In a first embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope including an amino acidsequence corresponding to an amino acid sequence of progastrin.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the N-terminal part of progastrin, wherein said amino acidsequence may include residues 10 to 14 of hPG, residues 9 to 14 of hPG,residues 4 to 10 of hPG, residues 2 to 10 of hPG or residues 2 to 14 ofhPG, wherein the amino acid sequence of hPG is SEQ ID NO 1.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the C-terminal part of progastrin, wherein said amino acidsequence may include residues 71 to 74 of hPG, residues 69 to 73 of hPG,residues 71 to 80 of hPG (SEQ ID NO 40), residues 76 to 80 of hPG, orresidues 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQID NO 1.

In a particular embodiment of a method for the in vitro diagnosis ofesophageal cancer according to the invention, said method comprises astep of contacting a biological sample from a subject with a firstmolecule which binds to a first part of progastrin and with a secondmolecule which binds to a second part of progastrin. In a moreparticular embodiment, wherein said progastrin-binding molecule is anantibody, a biological sample from a subject is contacted with anantibody which binds to a first epitope of progastrin and with a secondantibody which binds to a second epitope of progastrin.

In a preferred embodiment, the method of the present invention for thediagnosis of esophageal cancer comprises the detection of progastrin ina biological sample from a human subject.

In a more preferred embodiment, the method of the present invention forthe diagnosis of esophageal cancer comprises the detection of theconcentration of progastrin in a biological sample from a human subject.

In another particular embodiment, the method of the present inventionfor the diagnosis of esophageal cancer comprises the detection of theconcentration of progastrin in a biological sample from a human subject,wherein said biological sample is selected from blood, serum and plasma.

In a further preferred embodiment, the method of the present inventioncomprises contacting a sample from said subject with an anti-hPGantibody as described above, wherein the binding of said anti-hPGantibody in the sample indicates the presence of esophageal cancer insaid subject.

In a more particular embodiment, the method of the present inventioncomprises contacting sample from said subject with an anti-hPG antibodyas described above, wherein a concentration of progastrin superior to 10pM in said sample is indicative of the presence of esophageal cancer insaid subject.

More preferably, the method of the present invention comprisescontacting a sample from said subject with an anti-hPG antibody asdescribed above, wherein a concentration of progastrin superior to 10pM, 20 pM, 30 pM or 40 pM in said sample is indicative of the presenceof esophageal cancer in said subject.

Still more preferably, the method of the present invention comprisescontacting a sample from said subject with an anti-hPG antibody asdescribed above, wherein a concentration of progastrin superior to 10pM, 20 pM, 30 pM, 40 pM in said plasma is indicative of the presence ofmetastasized esophageal cancer in said subject

The present invention also relates to methods for monitoring theefficacy of a treatment for esophageal cancer in a patient, such aschemotherapy, biological therapy, immunotherapy or antibody therapy, bydetermining the concentration of progastrin in a first sample, such as abodily fluid or biopsy of esophageal cancer, obtained from a patientbefore treatment for esophageal cancer, and then comparing theconcentration of progastrin in the first sample to that in a secondsample obtained from the same patient after treatment, where a reductionin the concentration of progastrin in said second sample compared tosaid first sample indicates that the treatment was effective.

In a particular embodiment, a method according to the inventioncomprises comparing the concentration of progastrin in a biologicalsample obtained from a patient with a predetermined value ofconcentration of progastrin in the sample, in a more particularembodiment, said predetermined value is chosen among: an mean, oraverage, of sample values based on the mean, or average, determinationof the value in a population free of esophageal cancer, a progastrinconcentration value obtained when the patient was known to be free ofesophageal cancer.

In a particular embodiment, a method according to the invention for thein vitro diagnosis of esophageal cancer comprises the determination ofprogastrin concentration in a sample from said patient and a seconddiagnosis test of esophageal cancer. In a more particular embodiment, amethod according to the invention for the in vitro diagnosis ofesophageal cancer comprises the determination of progastrinconcentration in a sample from said patient and a second diagnosis testof esophageal cancer, wherein

In a particular embodiment of the invention, a method according to thepresent invention comprises the determination of the level of progastrinover time in samples from a patient who has been or is being treated foresophageal cancer.

In another aspect, the subject matter of the present invention relatesto a composition for use in the prevention or the treatment ofesophageal cancer, wherein said composition comprises aprogastrin-binding antibody, or an antigen-binding fragment thereof.

Antibody compositions for use in the methods of the invention can beprepared as different formulations, including, but not limited to, anaqueous suspension, for administration by a variety of routes,including, but not limited to, parenteral, intrathecal, subcutaneous,intravenous, intramuscular, intraperitoneal, infusion or bolusadministration. In some embodiments, the composition is formulated forparenteral administration, and in some specific embodiments, intravenousinjection by infusion.

In a particular embodiment, a composition for use in the prevention orthe treatment of esophageal cancer, according to the invention,comprises an effective dose the anti-progastrin antibodies of theinvention ranges from 0.001 mg/kg to about 250 mg/kg, which may be givenin one administration, or over multiple, spaced administrations.

In a particular embodiment, a composition for use in the prevention orthe treatment of esophageal cancer, according to the invention,comprises a progastrin-binding antibody, or an antigen-binding fragmentthereof selected among polyclonal antibodies, monoclonal antibodies,chimeric antibodies, single chain antibodies, camelized antibodies, IgA1antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies and IgMantibodies. Preferably, said antibodies are those described above. Morepreferably, said antibodies are humanized antibodies.

In a more particular embodiment, a composition for use in the preventionor the treatment of esophageal cancer, according to the invention,comprises a progastrin-binding antibody, or an antigen-binding fragmentthereof which has an affinity for progastrin of at least 5000 nM, atleast 500 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 5 pM, 1pM, or at least 0.1 pM, as determined by a method such asabove-described.

In an even more particular embodiment, a composition for use in theprevention or the treatment of esophageal cancer comprises aprogastrin-binding antibody, wherein said progastrin-binding molecule,or an antigen-binding fragment thereof, is a neutralizing antibody.

The expression “neutralizing anti-PG antibody” designates an antibodythat binds PG and blocks PG-dependent signaling, resulting in theinhibition of PG-induced responses in tumor cells, and particularly inesophageal tumor cells. Inhibiting PG-induced responses of esophagealcells may be mediated by repression of cell differentiation, repressionof cell death, and/or stimulation of cell proliferation.

In another particular embodiment, a composition for use in theprevention or the treatment of esophageal cancer comprises aprogastrin-binding antibody, wherein said progastrin-binding molecule,or an antigen-binding fragment thereof, is a humanized antibody.

In a particular embodiment, a composition for use in the prevention orthe treatment of esophageal cancer comprises a progastrin-bindingantibody, wherein said progastrin-binding molecule, or anantigen-binding fragment thereof, is conjugated to a cytotoxic molecule.

In another particular embodiment, a composition for use in theprevention or the treatment of esophageal cancer for a patient comprisesa progastrin-binding antibody, wherein said patient has been diagnosedwith esophageal cancer by a method according to the present invention,wherein a concentration of progastrin is higher in a biological samplefrom said patient than in a reference sample.

In a more particular aspect, the present invention relates to acomposition for use in the prevention or the treatment of esophagealcancer according to the invention, wherein said progastrin-bindingantibody, or an antigen-binding fragment thereof, is selected amongN-terminal anti-progastrin antibodies and C-terminal anti-progastrinantibodies.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising a composition for use in the prevention or thetreatment of esophageal cancer according to the invention, and apharmaceutically acceptable carrier. More specifically, thepharmaceutical composition for use in the prevention or the treatment ofesophageal cancer according to the invention, comprises an antibody asdescribed above and a pharmaceutically acceptable carrier.

In a more particular aspect, the present invention relates to apharmaceutical composition comprising a composition for use in theprevention or the treatment of esophageal cancer according to theinvention, and a pharmaceutically acceptable carrier, wherein saidanti-progastrin antibody is administered at a dose from 0.001 mg/kg to250 mg/kg, and preferably at a dose of at least 0.005 mg/kg, at least0.01 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg,at least 1 mg/kg, at least 5 mg/kg, at least 10 mg/kg, at least 50 mg/kgor at least 100 mg/kg. In another aspect, the present invention relatesto a kit of parts comprising a composition for use in the prevention orthe treatment of esophageal cancer, according to the invention, and ananti-cancer therapeutic molecule.

Indeed, treatment with anti-PG monoclonal antibodies as described hereincan be combined with, or adjunctive to, other therapy. Non-limitingexamples of other therapy include chemotherapeutic treatment, radiationtherapy, surgical resection, and antibody therapy.

In another aspect, the present invention relates to a kit of partcomprising a composition for use in the prevention or the treatment ofesophageal cancer, according to the invention, and an anti-cancertherapeutic molecule chosen among: a chemotherapeutic molecule, atargeted therapy molecule.

In a particular embodiment, the present invention relates to kits ofpart comprising, for the simultaneous, sequential or separateadministration, a composition for the treatment of esophageal canceraccording to the invention and a chemotherapeutic molecule. Usefulchemotherapeutic molecules for this purpose, include, but are notlimited to folate antagonists, purine antagonists, pyrimidineantagonists, DNA alkylating molecules, DNA cross-linking drugs,antibiotics, platinum complexes, proteasome inhibitors, mitotic spindlepoisons, topoisomerase inhibitors, tyrosine kinase inhibitors, andothers.

In another particular embodiment, the present invention relates to kitsof part comprising, for the simultaneous, sequential or separateadministration, a composition according to the invention and acomposition comprising another targeted therapy molecule. Such targetedtherapy molecule include, but are not limited to antibodies that targetEGFR, such as cetuximab or panitumumab, antibodies that target VEGF,such as bevacizumab, antibodies that target HER2, such as trastuzumab orpertuzumab, antibodies that target PD-1 and PDL-1, such aspembrolizumab, antibodies that target CTLA-4, such as ipilimumab, smallmolecule drugs that target EGFR, such as erlotinib, small molecule drugsthat target BRAF, such as vemurafenib or dabrafenib, a recombinantfusion protein that target VEGF, such as Aflibercept.

In another particular aspect, the present invention relates to the useof a progastrin-binding antibody, or an antigen-binding fragmentthereof, for the diagnosis of esophageal cancer.

In another particular aspect, the present invention relates to the useof a progastrin-binding antibody, or an antigen-binding fragmentthereof, for the prevention or the treatment of esophageal cancer.

In a more particular aspect, the present invention relates to the use ofa progastrin-binding antibody, or an antigen-binding fragment thereof,for the prevention or the treatment of esophageal cancer for a patient,wherein the concentration of progastrin in a biological sample of saidpatient has been determined and is higher than the concentration ofprogastrin of a reference biological sample.

In a more particular aspect, the present invention relates to the use ofa progastrin-binding antibody, or an antigen-binding fragment thereof,for the prevention or the treatment of esophageal cancer for a patient,wherein said patient presents metastasis.

In an even more particular aspect, the present invention relates to theuse of a progastrin-binding antibody, or an antigen-binding fragmentthereof, for the prevention or the treatment of esophageal cancer for apatient, wherein said patient presents metastasis and wherein theconcentration of progastrin in a biological sample of said patient hasbeen determined and is higher than the concentration of progastrin of areference biological sample.

The constituents of which the combination is composed may beadministered simultaneously, separately, or sequentially so as to obtainthe maximum efficacy of the combination; it being possible for eachadministration to vary in its duration from a rapid administration to acontinuous perfusion.

As used herein, “simultaneous administration” refers to theadministration of the two compounds of the composition according in asingle and unique pharmaceutical form. As used herein, “separateadministration” refers to the administration, at the same time, of thetwo compounds of the composition according to the invention in distinctpharmaceutical forms. As used herein, “sequential administration” refersto the successive administration of the two compounds of the compositionaccording to the invention, each in a distinct pharmaceutical form.

A “therapeutically effective amount”, as used herein, refers to theminimum concentration or amount of a compound (or of compounds), whichis effective to prevent, alleviate, reduce or ameliorate symptoms ofdisease or prolong the survival of the patient being treated.

The characteristics of the embodiments of the invention will becomefurther apparent from the following detailed description of examplesbelow.

FIGURE LEGEND

FIG. 1 : median plasmatic concentration of progastrin in esophagealcancer patients (n=12), and in control patients (n=103)—Mann Whitneytest two-tailed, *** p<0.001

FIG. 2 : Number of OE33 spheres formed following treatment with control(CT Hz) or anti-PG humanized antibody (PG Hz) under ultra-low adherentconditions—Two-tailed t-test, * p<0.05.

EXAMPLES Example 1: Detection of Plasmatic Progastrin ConcentrationUsing Polyclonal Antibodies

Plasma progastrin levels were quantified by ELISA through the use of twospecific anti-progastrin antibodies: capture antibodies are coated onthe wells of the plate, whereas revelation antibodies are used to detectprogastrin and mediates revelation of the signal.

In the present example, quantification is based on the ELISA methodwhich allows, through the use of a substrate whose reaction emits light,to assign a value proportional to the luminescence amount of antibodiesbound to the antigen retained by capture antibodies.

Material

Reagents and apparatus are listed in Table 7:

TABLE 7 Designation Provider Reference Plates MaxiSORP white Nunc, 96wells Dutscher # 055221 Sodium Carbonate/Bicarbonate Sigma # 21851 DPBS1X Lonza # P04- 36500 Tween-20 Biosolve # 20452335 BSA Euromedex # 04-100-810-C Streptavidin-HRP Pierce # 21130 (Thermo) SuperSignal ELISAFemto Maximum Pierce # 37074 Sensitivity Substrate (Thermo)Anti-ProGastrin Polyclonal Antibody Eurogentec /

Polyclonal antibodies were obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID NO 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID NO 40), according to standard protocols.

The binding characteristics of polyclonal antibodies against progastrinused in this assay are the following: absence of binding to G34-Gly,G34, G17-Gly, G17, binding to full length progastrin.

96 wells plates are coated by preparing a solution of carbonate—sodiumbicarbonate, 50 mM pH 9.6 by dissolving the contents of one capsule in100 ml of MilliQwater. A solution of capture antibody (3 μg/ml),corresponding to polyclonal antibodies obtained by using the C-terminalof progastrin FGRRSAEDEN (SEQ ID NO 40) is prepared in carbonate buffer.100 microliters of antibodies solution is added to each well andincubated at 4° C. for 16 hours (1 night). Plates are then blocked byeliminating the antibodies solution and wash 3 times with 300 μl1×PBS/0.1% TWEEN®, then adding 200 μl of blocking buffer (1×PBS/0.1%TWEEN®/0.1% BSA) per well, and incubated 2 hours at 22° C. Blockingbuffer is then eliminated, wells are washed 3 times with 300 μl1×PBS/0.1% TWEEN®.

Plasma dilution is performed as follows: The plasma is used pure,diluted ½, ⅕ and 1/10. Dilutions are prepared from pure plasma in1×PBS/0.1% Tween 20/0.1% BSA.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations were prepared as follows:

-   -   Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer    -   Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer    -   Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer    -   Solution D: 500 pM, 5,55 μl of C+494.5 μl of the diluent    -   Solution E: 250 pM, 250 μl of D+250 μl of the diluent    -   Solution F: 100 pM, 200 μl of E+300 μl of the diluent    -   Solution G: 50 pM, 250 μl of F+250 μl of the diluent    -   Solution H: 25 pM, 200 μl of G+200 μl of the diluent    -   Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

For the preparation of test samples, approximately 500 μl of each sampleare set aside and stored until analysis (and confirmation if necessary)of the results. 100 μl of each point of the range and/or plasmas areassayed pure, diluted to ½, ⅕ and 1/10, and incubated for 2 hours at 22°C. on the plates.

For the revelation of the test, the plates are washed 3 times with 300μl 1×PBS/0.1% TWEEN®. A solution of the polyclonal rabbitanti-progastrin antibody, wherein said antibodies have been obtained byusing the N-terminal part of progastrin as an immunogen, coupled tobiotin to 0.5 μg/ml, is prepared by dilution in 1×PBS/0.1% TWEEN®/0.1%BSA. 100 μl of this solution is added to each well. Incubation takesplace for 1 hour at 22° C. The revelation with streptavidin-HRP isperformed by removing detection antibody and wash 3 times with 300 μl1×PBS/0.1% TWEEN®, then preparing a solution of Streptavidin-HRP at 20ng/ml diluted in 1×PBS/0.1% TWEEN®/0.1% BSA, wherein 100 Add 100 μl ofthis solution is added to each well, before incubation for 1 hour at 22°C.

The detection consists of eliminating streptavidin-HRP and wash 3 timeswith 300 μl 1×PBS/0.1% TWEEN®, then adding 100 μl of chemiluminescentsubstrate solution per well. The substrate solution is prepared bymixing equal volumes of the two solutions SuperSignal ELISA Femto kit,20 ml+20 ml, 30 minutes before use and stored at room temperature in thedark. Luminescence is read after 5 minutes incubation at roomtemperature in the dark.

For each condition, the test is performed in triplicate and the resultsof the ranges will be presented as a graph showing the change inluminescence depending on the progastrin concentration. For each plasmadilution, the concentration of progastrin is determined using theequation of the linear regression line of the corresponding range (range1/10th for a sample diluted to 1/10th).

Methods and Results

The median plasmatic concentration of progastrin is 42.3 pM in patientshaving esophageal cancer (n=12), whereas the median plasmaticconcentration of progastrin is 0 pM in control patients (n=103) (FIG. 1). These data demonstrate that patients with esophageal cancer havehigher concentrations of progastrin in their plasma compared to healthycontrol individuals.

These data demonstrate that patients with esophageal cancer have higherlevels of progastrin in their plasma compared to healthy controlindividuals.

Example 2: Detection of Progastrin Concentration Using MonoclonalAnti-Progastrin Antibodies

The wells of Nunc MaxiSORP 96-well plates are coated with a firstprogastrin-specific antibody as follows. Anti-progastrin monoclonalantibodies specific for the carboxy-terminal region of progastrin arediluted to a concentration of 3 μg/ml in a solution of 50 mM, pH 9.6sodium carbonate/bicarbonate buffer in MilliQ water.

A total of 100 μl of the antibody solution is then added to each well ofthe 96-well plates, and incubated overnight at 4° C. After binding, theantibody solution is removed from the wells, which are then washed threetimes with 100 μl wash buffer (IX PBS/0.1% TWEEN®). A total of 100 μlblocking buffer (IX PBS/0.1% TWEEN®/0.1% BSA) is then added to each welland incubated for 2 hours at 22° C. Blocking buffer is then removed andthe wells washed three times with wash buffer. Plasma or serum samplesisolated from patients is then added to the wells in a volume of 100 μlin a dilution series, typically 1:1, 1:2, 1:5 and 1:10 dilutions, and isthen incubated for 2 hours at 22° C. Plasma or serum samples areanalyzed in duplicate.

Assays also include two standard curves. The first standard curve isprepared using dilutions of recombinant progastrin to a final amount of1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.01 ng, and 0 ng per well. Thesecond standard curve, which serves as a negative control, is preparedfrom progastrin-negative human serum diluted in blocking buffer at thesame dilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.Alternatively, when plasma samples are being assayed, the secondstandard curve, which serves as a negative control, is prepared fromprogastrin-negative human plasma diluted in blocking buffer at the samedilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.

After incubation with the plasma or serum samples is complete, the wellcontents are removed and the wells are washed three times with washbuffer, 100 μl/well, after which progastrin bound to the first antibodyis detected using a second antibody specific for progastrin, as follows.

Biotin-coupled anti-progastrin monoclonal antibodies specific for theamino-terminal region of progastrin are diluted in blocking buffer to aconcentration of 0.1 to 10 μl g/ml, depending on the antibody. A totalof 100 μl of the antibody solution is then added to each well, andincubated for 1 hour at 22° C.

After secondary antibody binding is complete, the plates are washedthree times with wash buffer, 100 μl/well, after which 100 μl of asolution of streptavidin-HRP (25 ng/ml in blocking buffer) is added toeach well and incubated for 1 hour at 22° C. After incubation with thestreptavidin-HRP solution is complete, the plates are washed three timeswith wash buffer, 100 μl/well. Thereafter, 100 μl of chemiluminescentsubstrate prepared using a Pierce SuperSignal ELISA Femto MaximumSensitivity Chemiluminescent Substrate kit, is added per well, incubatedfor 5 min at room temperature in the dark, and then read on aluminometer.

Based on the luminometer readings, linear regression analysis is used toderive the equation of the lines corresponding to the standard curvedata. Using this equation, the concentration of progastrin in thevarious patient samples is then calculated.

The median plasmatic concentration of progastrin is calculated inpatients having esophageal cancer and compared to the median plasmaticconcentration of progastrin in plasma of control patients. These datademonstrate that patients with esophageal cancer had elevated levels ofprogastrin in their plasma compared to healthy control individuals.

Example 3: Neutralizing Activity of Anti-hPG Antibodies on Cancer CellLines

3.1. Neutralizing Activity of Anti-hPG Monoclonal Antibodies

TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 are cell lines commonlyused to study esophageal cancer, which produce and secrete progastrin.Monoclonal antibodies to PG are tested for their ability to inhibitproliferation in these different cell lines. Survival of cells from eachTE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 cell line is tested usingdifferent anti-hPG monoclonal antibodies.

For each experiment, 50,000 cells are seeded into 6-well plates inmedium containing fetal calf serum and incubated for 8 hours. Cells areserum-starved overnight, and starting at 24 hours after seeding (time“TO”), cells are treated in sextuplicates every 12 h for 48 hours, inthe absence of fetal calf serum, with 1 to 20 μg/ml of monoclonalcontrol antibodies (monoclonal antibody anti-puromycin)(CT mAb), or with1 to 20 μg/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPGmonoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

Said mAb is a C-terminal anti-hPG antibody, selected among:

-   -   An antibody comprising a heavy chain comprising CDR-H1, CDR-H2        and CDR-H3 of amino acid sequences SEQ ID NO 28, 29 and 30, and        a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid        sequences SEQ ID NO 31, 32 and 33,    -   An antibody comprising a heavy chain comprising CDR-H1, CDR-H2        and CDR-H3 of amino acid sequences SEQ ID NO 34, 35 and 36, and        a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid        sequences SEQ ID NO 37, 38 and 39.

or a N-terminal anti-hPG antibody selected among:

-   -   An monoclonal antibody comprising a heavy chain comprising        CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO 4, 5        and 6, respectively, and a light chain comprising CDR-L1, CDR-L2        and CDR-L3 of amino acid sequences SEQ ID NO 7, 8 and 9,    -   An antibody comprising a heavy chain comprising CDR-H1, CDR-H2        and CDR-H3 of amino acid sequences SEQ ID NO 10, 11 and 12,        respectively, and a light chain comprising CDR-L1, CDR-L2 and        CDR-L3 of amino acid sequences SEQ ID NO 13, 14 and 15,        respectively,    -   An antibody comprising a heavy chain comprising CDR-H1, CDR-H2        and CDR-H3 of amino acid sequences SEQ ID NO 16, 17 and 18,        respectively, and a light chain comprising CDR-L1, CDR-L2 and        CDR-L3 of amino acid sequences SEQ ID NO 19, 20 and 21,        respectively,    -   An antibody comprising a heavy chain comprising CDR-H1, CDR-H2        and CDR-H3 of amino acid sequences SEQ ID NO 22, 23 and 24,        respectively, and a light chain comprising CDR-L1, CDR-L2 and        CDR-L3 of amino acid sequences SEQ ID NO 25, 26 and 27,        respectively,

The number of cells at TO is counted in a control well, for eachexperiment.

Specifically, the number of live cells in both control and anti-hPG mAbtreated wells is counted at 48 hours, then the difference between eachcell count and the cell count determined at TO, is calculated. Theresulting number of anti-hPG mAb-treated cells is then expressed as apercentage of the number of control mAb-treated cells.

Treatment with anti-hPG monoclonal antibodies reduces cell number ascompared to treatment with control antibody. Statistical significance isdetermined using a one-way ANOVA with a Tukey post-hoc test: *=p<0.05,**=p<0.01, and ***=p<0.001. In each cell line, anti-hPG antibodiesreduce cell survival.

3.2. Neutralizing Activity of Anti-hPG Humanized Antibodies on CellSurvival

Humanized antibodies to PG are tested for their ability to inhibitproliferation of TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 celllines. Survival of cells from each TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19and OE33 cell line is tested using different anti-hPG humanizedantibodies.

For each experiment, 50,000 cells are seeded into 6-well plates inmedium containing fetal calf serum and incubated for 8 hours. Cells areserum-starved overnight, and starting at 24 hours after seeding (time“TO”), cells are treated in sextuplicates every 12 h for 48 hours, inthe absence of fetal calf serum, with 1 to 20 μg/ml of humanized controlantibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20μg/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanizedantibody or a N-terminal anti-hPG humanized antibody. The number ofcells at TO is counted in a control well, for each experiment.

Specifically, the number of live cells in both control and anti-hPG Hztreated wells is counted at 48 hours, then the difference between eachcell count and the cell count determined at TO, is calculated. Theresulting number of anti-hPG Hz-treated cells is then expressed as apercentage of the number of control mAb-treated cells.

Treatment with anti-hPG Hz antibodies reduces cell number as compared totreatment with control antibody. Statistical significance is determinedusing a one-way ANOVA with a Tukey post-hoc test: *=p<0.05, **=p<0.01,and ***=p<0.001. In each cell line, anti-hPG antibodies reduce cellsurvival.

3.3. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on CancerStem Cell Frequency

Monoclonal antibodies to PG are tested for their ability to reducecancer stem cell (CSC) frequency in TE-1, TE-4, TE-6, KYSE30, FLO-1,OE19 and OE33 cell lines using Extreme Limiting Dilution Assay (ELDA).CSC frequency from each TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33cell line is tested using different anti-hPG monoclonal antibodies.

For each experiment, cells are seeded in ultra-low attachment (ULA) P96(96-well plates) at fixed cellular concentrations per well using a FACSAria flow cytometer, and a range of concentrations is used from one to500 cells per well. The cells are cultivated for up to 11 days in ULAplates with M11 medium (Macari et al, Oncogene, 2015) and treated every3 or 4 days with 1 to 20 μg/ml of monoclonal control antibodies(monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 μg/mlanti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonalantibody or a N-terminal anti-hPG monoclonal antibody, as disclosed inExample 3.1.

Specifically, at the end of the incubation phase, the plates areobserved with a phase-contrast microscope and the number of positivewells per cellular concentration is assessed. Finally, the ELDA webtoolis used to calculate the CSC frequencies of each treatment group andtest for any statistical difference between groups (modified Chi-squaretest).

Treatment with anti-hPG monoclonal antibodies reduces CSC frequency ascompared to treatment with control antibody.

3.4. Neutralizing Activity of Anti-hPG Humanized Antibodies on CancerStem Cell Frequency

-   -   Sphere formation assay

Humanized antibodies to PG are tested for their ability to reduce cancerstem cell (CSC) frequency in FLO-1, OE19 and OE33 cell lines usingsphere formation assay.

For each experiment, 200 cells are seeded in 24-well ultra-lowattachment (ULA). The cells are cultivated for up to 10 days in ULAplates with M11 medium (Macari et al, Oncogene, 2015) and treated every3 or 4 days with 20 μg/ml of humanized control antibodies (anti-humanFcG1, from BioXCell)(CT Hz), or with 20 μg/ml anti-hPG Hz (PG Hz),wherein said Hz is a C-terminal anti-hPG humanized antibody or aN-terminal anti-hPG humanized antibody.

Specifically, at the end of the incubation phase, the wells arephotographed via brightfield microscopy, the pictures are analyzed andthe spheres with a mean diameter above 25 μm are counted.

Treatment with anti-hPG humanized antibodies reduces CSC frequency ascompared to treatment with control antibody.

-   -   Extreme Limiting Dilution Assay

Humanized antibodies to PG are tested for their ability to reduce cancerstem cell (CSC) frequency in TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 andOE33 cell lines using Extreme Limiting Dilution Assay (ELDA). CSCfrequency from each TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 cellline is tested using different anti-hPG humanized antibodies.

For each experiment, cells are seeded in ultra-low attachment (ULA) P96(96-well plates) at fixed cellular concentrations per well using a FACSAria flow cytometer, and a range of concentrations is used from one to500 cells per well. The cells are cultivated for up to 11 days in ULAplates with M11 medium (Macari et al, Oncogene, 2015) and treated every3 or 4 days with 1 to 20 μg/ml of humanized control antibodies(anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 μg/ml anti-hPGHz, wherein said Hz is a C-terminal anti-hPG humanized antibody or aN-terminal anti-hPG humanized antibody.

Specifically, at the end of the incubation phase, the plates areobserved with a phase-contrast microscope and the number of positivewells per cellular concentration is assessed. Finally, the ELDA webtoolis used to calculate the CSC frequencies of each treatment group andtest for any statistical difference between groups (modified Chi-squaretest).

Treatment with anti-hPG humanized antibodies reduces CSC frequency ascompared to treatment with control antibody.

3.5. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on theWNT/β-Catenin Pathway

TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 are cell lines commonlyused to study esophageal cancer, which produce and secrete progastrin.Monoclonal antibodies to PG were tested for their ability to inhibit theWNT/β-catenin pathway in these different cell lines using the expressionof the protein survivin, a well-known WNT/β-catenin pathway targetedgene, as read-out. Survivin expression from each TE-1, TE-4, TE-6 andKYSE30 cell line is tested using different anti-hPG monoclonalantibodies.

For each experiment, 50,000 cells are seeded into 6-well plates inmedium containing fetal calf serum and incubated for 8 hours. Cells areserum-starved overnight, and starting 24 hours after seeding cells aretreated in quadruplicate every 12 h for 72 hours, in the absence offetal calf serum, with 1 to 20 μg/ml of monoclonal control antibodies(monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 μg/mlanti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonalantibody or a N-terminal anti-hPG monoclonal antibody.

Specifically, after 72 hours of treatment, cells are harvested and totalproteins are extracted using RIPA buffer. An equal amount of proteinfrom CT mAb or anti-hPG mAb treated cells are then subjected to awestern blot using anti-survivin antibody (monoclonal antibody, #2802from Cell Signaling) and anti-actin antibody as loading control(monoclonal antibody, #A4700 from SIGMA). Quantification is performedusing the GBOX chemi system from Syngene.

Treatment with anti-hPG monoclonal antibodies reduces survivinexpression as compared to treatment with control antibody. Statisticalsignificance is determined using a unpaired Student's T-test: *=p<0.05,**=p<0.01, and ***=p<0.001.

3.6. Neutralizing Activity of Anti-hPG Humanized Antibodies on theWNT/β-Catenin Pathway

Humanized antibodies to PG are tested for their ability to inhibit theWNT/β-catenin pathway in TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33cell lines using the expression of the protein survivin, a well-knownWNT/β-catenin pathway targeted gene, as read-out. Survivin expressionfrom each TE-1, TE-4, TE-6, KYSE30, FLO-1, OE19 and OE33 cell line istested using different anti-hPG humanized antibodies.

For each experiment, 50,000 cells are seeded into 6-well plates inmedium containing fetal calf serum and incubated for 8 hours. Cells areserum-starved overnight, and starting 24 hours after seeding cells aretreated in quadruplicate every 12 h for 72 hours, in the absence offetal calf serum, with 1 to 20 μg/ml of humanized control antibodies(anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 μg/ml anti-hPGHz, wherein said Hz is a C-terminal anti-hPG humanized antibody or aN-terminal anti-hPG humanized antibody.

Specifically, after 72 hours of treatment, cells are harvested and totalproteins are extracted using RIPA buffer. An equal amount of proteinfrom CT Hz or anti-hPG Hz treated cells are then subjected to a westernblot using anti-survivin antibody (monoclonal antibody, #2802 from CellSignaling) and anti-actin antibody as loading control (monoclonalantibody, #A4700 from SIGMA). Quantification is performed using the GBOXchemi system from Syngene.

Treatment with anti-hPG humanized antibodies reduces survivin expressionas compared to treatment with control antibody. Statistical significanceis determined using a unpaired Student's T-test: *=p<0.05, **=p<0.01,and ***=p<0.001.

BIBLIOGRAPHIC REFERENCE

-   Kaz et al, Cancer Letters, 2014 Jan. 28; 342(2):193-9. “Epigenetic    biomarkers in esophageal cancer.”

The invention claimed is:
 1. A method for treating esophageal cancer ina patient in need thereof, said method comprising administering acomposition comprising a monoclonal progastrin-binding antibody, or anantigen-binding fragment thereof, to said patient, wherein saidmonoclonal progastrin-binding antibody or an antigen-binding fragmentthereof binds progastrin, but does not bind gastrin-17 (G17), gastrin-34(G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extendedgastrin-34 (G34-Gly), wherein said antigen-binding fragment thereofcomprises the 6 complementary determining regions (CDR) of themonoclonal progastrin-binding antibody from which it is derived andwherein said CDRs are defined by international ImMungoGenTicsinformation system (IMGT), and wherein said monoclonalprogastrin-binding antibody is selected from the group consisting of: anantibody comprising a heavy chain (H) comprising CDR-H1, CDR-H2 andCDR-H3 comprising amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6, respectively, and a light chain (L) comprising CDR-L1, CDR-L2and CDR-L3 comprising amino acid sequences SEQ ID NO:7, SEQ ID NO:8, andSEQ ID NO:9, respectively, an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, anantibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3comprising amino acid sequences SEQ ID NO:16, SEQ ID NO:17, and SEQ IDNO:18, respectively, and a light chain comprising CDR-L1, CDR-L2 andCDR-L3 comprising amino acid sequences SEQ ID NO:19, SEQ ID NO:20, andSEQ ID NO:21, respectively, an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, anantibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3comprising amino acid sequences SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30, respectively, and a light chain comprising CDR-L1, CDR-L2 andCDR-L3 comprising amino acid sequences SEQ ID NO:31, SEQ ID NO:32, andSEQ ID NO:33, respectively, and an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. 2.The method of claim 1, wherein said monoclonal progastrin-bindingantibody, or antigen-binding fragment thereof, is selected from thegroup consisting of humanized antibodies, single chain antibodies,immunoglobulin (Ig) A1 (IgA1) antibodies, IgA2 antibodies, IgDantibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3antibodies, IgG4 antibodies, and IgM antibodies.
 3. The method of claim1, wherein said monoclonal progastrin-binding antibody is a humanizedantibody.
 4. The method of claim 1, wherein said monoclonalprogastrin-binding antibody, or an antigen-binding fragment thereof, isconjugated to a cytotoxicmolecule.
 5. The method of claim 1, whereinsaid esophageal cancer is metastatic.
 6. The method of claim 1, furthercomprising the simultaneous, sequential, or separate administration of achemotherapeutic molecule selected from the group consisting of folateantagonists, purine antagonists, pyrimidine antagonists, DNA alkylatingmolecules, DNA cross-linking drugs, antibiotics, platinum complexes,proteasome inhibitors, mitotic spindle poisons, topoisomeraseinhibitors, and tyrosine kinase inhibitors.
 7. The method of claim 1,further comprising the simultaneous, sequential, or separateadministration of a targeted therapy molecule selected from the groupconsisting of antibodies that target epidermal growth factor receptor(EGFR), antibodies that target vascular endothelial growth factor(VEGF), antibodies that target human epidermal growth factor receptor(HER2), antibodies that target programmed cell death protein 1 (PD-1)and programmed death-ligand 1 (PDL-1), antibodies that target cytotoxicT-lymphocyte-associated protein 4 (CTLA-4), small molecule drugs thattarget EGFR, small molecule drugs that target B-Raf Proto-OncogeneSerine/Threonine Kinase (BRAF), and recombinant fusion proteins thattarget VEGF.
 8. The method of claim 7, wherein said antibody thattargets EGFR is cetuximab or panitumumab, wherein said antibody thattargets VEGF is bevacizumab, wherein said antibody that targets HER2 istrastuzumab or pertuzumab, wherein said antibody that targets PD-1 andPDL-1 is pembrolizumab, wherein said antibody that targets CTLA-4 isipilimumab, wherein said small molecule drug that targets EGFR iserlotinib, wherein said small molecule drug that targets BRAF isvemurafenib or dabrafenib, and wherein said recombinant fusion proteinthat targets VEGF is Aflibercept.
 9. A method for treating esophagealcancer in a patient in need thereof, said method comprisingadministering a pharmaceutical composition comprising a monoclonalprogastrin-binding antibody, or an antigen-binding fragment thereof, anda pharmaceutically acceptable carrier, to said patient, wherein saidmonoclonal progastrin-binding antibody or an antigen-binding fragmentthereof binds progastrin, but does not bind gastrin-17 (G17), gastrin-34(G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extendedgastrin-34 (G34-Gly), wherein said antigen-binding fragment thereofcomprises the 6 complementary determining regions (CDR) of themonoclonal progastrin-binding antibody from which it is derived andwherein said CDRs are defined by the international ImMungoGenTicsinformation system (IMGT), and wherein said monoclonalprogastrin-binding antibody is selected from the group consisting of: anantibody comprising a heavy chain (H) comprising CDR-H1, CDR-H2 andCDR-H3 comprising amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6, respectively, and a light chain (L) comprising CDR-L1, CDR-L2and CDR-L3 comprising amino acid sequences SEQ ID NO:7, SEQ ID NO:8, andSEQ ID NO:9, respectively, an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, anantibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3comprising amino acid sequences SEQ ID NO:16, SEQ ID NO:17, and SEQ IDNO:18, respectively, and a light chain comprising CDR-L1, CDR-L2 andCDR-L3 comprising amino acid sequences SEQ ID NO:19, SEQ ID NO:20, andSEQ ID NO:21, respectively, an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, anantibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3comprising amino acid sequences SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30, respectively, and a light chain comprising CDR-L1, CDR-L2 andCDR-L3 comprising amino acid sequences SEQ ID NO:31, SEQ ID NO:32, andSEQ ID NO:33, respectively, and an antibody comprising a heavy chaincomprising CDR-H1, CDR-H2 and CDR-H3 comprising amino acid sequences SEQID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and a lightchain comprising CDR-L1, CDR-L2 and CDR-L3 comprising amino acidsequences SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.